Prostaglandins are potent mediators of a wide range of physiological processes, including inflammation. Cyclooxygenase (COX) enzymes, also referred to as prostaglandin-endoperoxide synthases (PTGS), catalyze the rate limiting step in prostaglandin synthesis. Of the two cyclooxygenase enzymes, COX-2 enzyme (PTGS2) is inducible; it is produced in response to specific homeostatic changes. The discovery that COX-2 regulates the production of prostaglandins involved in the inflammation process, lead to the development of a class of agents that function as COX-2 inhibitors. Indeed, many anti-inflammatory agents now in the modern pharmacopoeia, including COX-2 inhibitors, have their origin in the practices of traditional medicine where plants and their extracts were administered to provide relief from pain, fever and inflammation. For example, the discovery that the active analgesic ingredients in Willow bark are salicylates, lead to the syntheses of acetylsalicylic acid (aspirin) and numerous other non-steroidal anti-inflammatory drugs (NSAIDs). Oral administration of these compounds is widely known to produce negative sequelae including irritation of the GI tract and bleeding. There has been, and remains a need, for efficacious topical anti-inflammatory compounds. With increased awareness of the potency of naturally-derived active agents, the need for such compounds is even more strongly felt.
The present invention is directed to a novel proteinaceous polysaccharide extract of Coriolus versicolor, a mushroom with a history both in traditional Chinese botanical remedies as well as in modern biomedicine as an adjunct in cancer treatments. As described in further detail below, the compositions of the present invention are made by a heretofore unknown process in which proteinaceous polysaccharides extracted from the fruiting body of C. versicolor are solubilized in an aqueous solvent and then hydrolyzed with a specific acid protease enzyme derived from R. miehei, thus forming an extract with novel anti-inflammatory properties—specifically, the ability to reduce the expression of mRNA coding for PTGS2 by at least about 67% as measured by DNA microarray analysis using a full-thickness epidermal skin tissue model.
Two peptidic polysaccharides derived from Coriolus versicolor have been widely-studied for their antiproliferative and antitumor activities. The two protein-bound polysaccharides, designated as PSK and PSP, are isolated from the CM-101 and COV-1 strains of C. versicolor, respectively. PSP and PSK are chemically similar—both have molecular weights of approximately 100 kDa; both have glutamic acid and aspartic acid as the two most abundant amino acid residues; and both have monosaccharides with α-1,4 and β-1,3 glucosidic linkages. The two compounds differ in that PSK contains fucose, while PSP contains rhamnose and arabinose. See, TB Ng. “A review of research on the protein-bound polysaccharide (polysaccharo-peptide, PSP) from the mushroom Coriolus versicolor (Basidiomycetes: Polyporaceae)” Gen Pharmacol Vol. 30, pp. 1-4 (1998).
PSK and PSP have also reported in the literature to have immuno-stimulatory effects. See TB Ng (1998) supra; see also Y. Dong, et al. “Antitumor effects of a refined polysaccharide peptide fraction isolated from Coriolus versicolor: in vitro and in vivo studies.” Res. Commun. Mol. Pathol. Pharmacol. Vol. 92, pp. 140-148 (1996). PSK is commercially available under the tradename Krestin from Sankyo Co. Ltd. (Tokyo, Japan). See also, K K Chu, S S Ho and A H Chow, J. Clin. Pharmacol. Vol. 42, pp. 976-984 (2002)(describing traditional usage, pharmacological activities, clinical effects, adverse and reactions, active constituents of C. versicolor, J. Cui and Y. Chisti, Biotechnology Advances, Vol. 21, pp. 109-122 (review of the physiological activity, uses and methods for producing polysaccharopeptides from C. versicolor).
While the prior art patent literature describes a number of processes for preparing polysaccharide polypeptide fractions from C. versicolor, these references neither teach nor suggest the methods for producing the compounds of the present invention, nor the use of the product of this novel process (i.e., as a potent topical anti-inflammatory agent).
U.S. Pat. No. 4,051,314 is directed to oral administration of deproteinized polysaccharide hydroxylate derived from C. versicolor that produces anticarcinogenic activity in mice. Compositions claimed in the present invention are proteinaceous hydrolyzates.
U.S. Pat. No. 4,761,402 teaches use of PSK as a starting material for the preparation of a lyophilized soluble phosphorylated glucan that can be topically applied to treat infectious disease in animals or humans. The '402 patent describes a soluble phosphorylated glucan prepared by a process comprising the steps of denaturing the three dimensional structure of a particulate glucan derived from C. versicolor followed by phosphorylation (mixing with phosphoric acid at a temperature of about 100° C. for several hours).
U.S. Pat. No. 4,818,752 describes the use of soluble phosphorylated glucan from C. versicolor prepared by the same process described in the '402 patent and its use in treating malignant neoplastic disease in animals and humans.
U.S. Pat. No. 4,833,131 describes a soluble phosphorylated glucan from C. versicolor prepared by the same process described in the '402 patent and its use in treating dermal wounds in animals and humans.
U.S. Pat. No. 5,374,714 describes a method of obtaining purified polypeptides from the COV-1 strain of C. versicolor by a specific process—High Performance Liquid Chromatography (HPLC) using an acid solvent including KCl in a reversed-phase column after extracting the materials from the first and last chromatograph peaks. The '714 patent describes purified polypeptides from this process as having cytotoxic effects on human tumor cell lines as well as immunopotentiating effects, namely increasing white blood cells, T and B lymphocytes, and IgG. The compounds of the present invention are made by a process different than described in the '714 patent, including a different starting material. The '714 patent does not suggest dermal application of the specified polypeptides, nor use as topical agent that reduces the appearance of chronological or environmentally caused aging.
U.S. Pat. Nos. 6,087,335 and 5,824,648 are related to the '714 patent and claim polypetides derived from the COV-1 strain of C. versicolor having a specific partial amino acid sequence, (SEQ ID NO: 1).
U.S. Pat. No. 4,202,969 describes low molecular weight (less than 5,000 kDa) nitrogen-containing polysaccharide compounds having anti-tumor activity extracted from C. versicolor by using a dilute alkaline solvent.
U.S. Pat. No. 4,851,395 describes a nitrogen-containing polysaccharide substantially free of units having a molecular weight below about 5,000 kDa produced by extracting C. versicolor with an aqueous alkaline solution (0.01 to 2.0N) at a temperature of from 50° C. to 100° C., neutralizing the resultant extract, followed by ultrafiltration or reverse osmosis to remove substantially all polymer units having a molecular weight below about 5,000 kDa.
U.S. Pat. Nos. 4,877,777, 4,900,722, and 4,975,421 assigned to BioGlucans, LP describe processes for preparing phosphorylated glucans from approximately two dozen source materials, including C. versicolor, in which the poly-[β-(1-3)glucopryanose] chains are phosphorylated in varying degrees by treating neutral glucans with phosphoric acid.
US Patent Application 2004/0137012 describes a pharmaceutically active agent having a molecular weight of from about 5,000 to about 20,000 useful in the treatment of diabetes where the agent is the product of hydrolysis of β(1→3)glucan derived from vegetable material, including Coriolus. 
U.S. Pat. No. 4,614,733 discloses polysaccharides derived from C. versicolor having specific characteristics, including, molecular weight (from 5,000 to 300,000), infrared and nuclear magnetic resonance absorption spectra and solubility profile (soluble in water but insoluble in pyridine, chloroform, and hexane). The polysaccharides taught in the '733 patent are made by a process comprising the steps of (i) extracting mycelia and fruit bodies of a basidiomycetous fungus, including C. versicolor, with an aqueous solvent, (ii) removing substances with a molecular weight of less than 5,000 by ultrafiltration and/or reverse osmosis; (iii) saturating the extract solution with ammonium sulfate; (iv) collecting the resultant precipitate, dissolving the precipitate in water, and desalting same; (v) passing the desalted solution through a column packed with an ion exchanger; (vi) concentrating and drying the solution to obtain the claimed polysaccharide.
German Patent DE 2,731,570 and related U.S. Pat. No. 4,140,578 describe nitrogen-containing polysaccharides having anti-cancer properties produced by a multi-step process involving cultivating a fungus from the genus Coriolus in an aqueous culture medium by submerged cultivation, drying the culture media together with the mycelia, producing an extract from the dried substance with water or an aqueous alkaline solution followed by ultrafiltration or reverse osmosis to remove substances having a molecular weight of less than 5,000.
Japanese Patent JP 52,083,996 and related U.S. Pat. Nos. 4,289,688 and 4,271,151 describe oral and intraperitoneal administration of protein-bound polysaccharides having an antitumor effect. The protein-bound polysaccharides are obtained by a process comprising the steps of concentrating an extract obtained from the mycelia and/or fruit bodies of a species of fungi belonging to the genus Coriolus in an aqueous solution, performing consecutive ammonium sulfate precipitations, followed by dialysis to remove salts, and finally spray-drying the desalted solution to obtain the claimed protein-bound polysaccharides.
Japanese Patent JP 09,309,842 describes an antitumor glycoprotein containing β-1,3-glucan in an amount of less than 0.001 mg/g with a molecular weight of 5,000-1,000,000 kDa from an extract of hyphae and fruiting body of Basidiomycota belonging to the genus Coriolus. The extract is obtaining by oxidation with periodic acid or its salt, followed by addition of a reducing agent, removal of low molecular compounds, and addition of β-1,3-glucanase.
Japanese Patent JP 49,048,896 describes soluble phosphorylated glucans from C. versicolor having a phosphorylated poly-[β-(1-3)glucopyranose] chain and its use in dermal wound healing, specifically as an agent impregnated into bandage, suture or dressing.
U.S. Pat. No. 7,048,932 describes compositions and methods for stimulating the immune system comprising administering a purified extract of C. versicolor having a molecular weight of 0.3 kDa to 5 kDa as determined by size exclusion chromatography and comprising at least one peptide-linked glucan in which the glucose molecules of the glucan are linked by a (1→3) linkage. The purified extract taught in the '932 patent is prepared by the steps of (i) treating C. versicolor with alkali; (ii) separating a supernatant; (iii) subjecting the supernatant to (a) cationic exchange followed by (b) anionic exchange; and (iv) collecting a fraction comprising the peptide-linked glucan.
A series of related patents assigned to Active Organics, LP—U.S. Pat. Nos. 5,976,556; 6,569,437; and 6,656,701—describe the uses of one or more acid protease enzymes in combination with an acidic buffering system that enhances epidermal exfoliation and/or epidermal cell renewal, thereby improving the texture or appearance of the skin.
Extracts of Rhizomucor miehei are commercially-available from a number of sources, including Novozymes, Inc. (Franklinton, N.C.), Valley Research (South Bend, Ind.) and Active Organics LP (Lewisville, Tex.), exhibit enzymatic activity, principally from acid proteases.
US Patent Application Publication No. 2007/0160563 discloses topical compositions comprising extracts of R. miehei that are substantially devoid of acid-protease activity and their use in treating dermatologic conditions, including reducing the appearance of signs of skin aging.